Table 1. Antifungal Breakpoints for Candida. 2,3. Agent S SDD or I R. Fluconazole < 8.0 mg/ml mg/ml. > 64 mg/ml.

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1 AUSTRALIAN ANTIFUNGAL SUSCEPTIBILITY DATA Part 1: The Yeasts In this article, an update of recent changes to the CLSI antifungal standards for susceptibility testing of yeasts is presented. We also summarise current Australian antifungal susceptibility data for Candida and Cryptococcus. A. CLSI M27-A3 and M27-S3 broth dilution standard for yeasts 1,2 The M27-A3 (most current) standard and M27-S3 informational supplement is intended for testing yeasts including Candida species and Cryptococcus species (C. neoformans and C. gattii) by using either a macro or micro broth dilution test system. 1. The M27-A3 standard recommends the use of RPMI-1640 medium supplemented with 0.2% glucose and buffered to a ph of 7.0 with mol/l MOPS. However, at the SA Pathology Mycology Unit, we routinely increase the glucose concentration to 20 g/l as documented in the M27-S3 supplement, which allows this modification to simplify endpoint determination. 2 This modification enhances the growth of Cryptococcus species and other yeasts that do not readily grow on standard RPMI medium. This modified RPMI glucose broth is now available from Oxoid Australia (TM 4040) in pre-packed 11 ml aliquots ready to use with the Sensititre YeastOne microbroth dilution plates. 2. The M27-A3 standard further recommends the application of less stringent MIC end points for the azoles and the echinocandins (50% inhibition) to improve interlaboratory agreement and also discriminate between putatively susceptible and resistant isolates. 2 Endpoint readings for amphotericin B remains at 100% inhibition. 3. Interpretation of results: For the most part, amphotericin B MICs for Candida species cluster between 0.25 and 1.0 μg/ml. However it must be stressed that the M27-A3 standard does not consistently permit detection of resistant strains and isolates with MICs of >1.0 μg/ml should be considered to be likely resistant. No MIC breakpoints for amphotericin B are recognized by the CLSI. However, interpretative breakpoints for Candida against fluconazole, itraconazole, voriconazole, 5-fluorocytosine and the echinocandins have been established (Table 1). 2,3 These breakpoints have also been used for isolates of Cryptococcus where there is some correlation between elevated MIC and treatment failure. 4 However the following caveats for fluconazole should now be noted 2 : (1) The 8 μg/ml susceptibility cutoff for fluconazole is not known with certainty. (2) The data would permit selection of either 4 or 8 μg/ml as the cutoff. (3) All C. glabrata isolates with an MIC <32 μg/ml should be reported as SDD. All C. krusei isolates should be reported as resistant (regardless of the MIC result). 2 Table 1. Antifungal Breakpoints for Candida. 2,3 Agent S SDD or I R Fluconazole < 8.0 mg/ml mg/ml. > 64 mg/ml. Itraconazole < mg/ml mg/ml. > 1 mg/ml. Voriconazole < 1 mg/ml 2 mg/ml. > 4 mg/ml Posaconazole* no break points set < 1 mg/ml likely to be susceptible Echinocandins < 2 mg/ml > 2 mg/ml not susceptible 5-Fluorocytosine < 4 mg/ml mg/ml. > 32 mg/ml. Amphotericin B* < 1 mg/ml 2 mg/ml. > 4 mg/ml *Tests for Amphotericin B maybe unreliable on RPMI media; CLSI provides no break point but <1 μg/ml likely to be susceptible. ASA Newsletter, August

2 4. Trailing ends points remain problematic, particularly for fluconazole and the echinocandins. To help resolve this issue the M27-S3 supplement has provided both 24 and 48 hour microdilution MIC ranges for the two QC strains and 11 systemic antifungal agents. 1,2 Ideally plates should be read at 24 hours whenever there is sufficient growth. Sensititre YeastOne YO10 Test Panel (manufactured by TREK but supplied in Australia by Dutec Diagnostics). This is a microtitre broth dilution method based on the CLSI standards described above for the testing of Candida, Cryptococcus and Aspergillus species. Each test consists of a disposable microtitre plate, which contains dried serial dilutions of nine antifungal agents, anidulafungin, caspofungin, micafungin, 5-Fluorocytosine, voriconazole, posaconazole, itraconazole, fluconazole and amphotericin B. The wells also contain Alamar Blue as a colorimetric indicator, which greatly improves the end point readability by a colour change from blue to pink. Results are expressed as an MIC and comparative studies against the CLSI method have shown favorable results. 5,6 Overall, the Sensititre YeastOne is a robust and reproducible test, easy to set up and the end points are usually clearly visible. It has excellent shelf life and the test also works with moulds, especially those that sporulate freely like Aspergillus. Australian antifungal susceptibility data: Candida and Cryptococcus ( ) Isolates recovered from patients with invasive candidaisis and cryptococcosis were tested for antifungal susceptibility with the CLSI M27-A3 microbroth susceptibility standardsfor yeasts. The MICs at which 90% (MIC 90 ) of the strains that were tested were inhibited, and the range of MICs, for major Candida spp. for common antifungal agents are summarised below. Table 2a: Echinocandin MICs: Candida spp. (echinocandins are not active against Cryptococcus). Anidulafungin Caspofungin Micafungin C. albicans (200) C. glabrata (194) C. parapsilosis (69) C. tropicalis (22) C. krusei (21) C. dubliniensis (12) other Candida sp (14) ASA Newsletter, August 2010

3 Table 2b. Fluconazole, voriconazole and posaconazole MICs: Candida and Cryptococcus spp. Fluconazole Voriconazole Posaconazole C. albicans (362) C. glabrata (303) C. parapsilosis (137) C. tropicalis (67) C. krusei (53) C. neoformans (46) C. gattii (23) Table 2c. Amphotericin B, 5-Fluorocytosine and itraconazole MICs: Candida and Cryptococcus spp. Amphotericin B 5-Fluorocytosine Itraconazole C. albicans (362) C. glabrata (303) C. parapsilosis (137) C. tropicalis (67) C. krusei (53) C. neoformans (46) C. gattii (23) All isolates of C. albicans, C. tropicalis and C.krusei were susceptible to all three echinocandins. Only two isolates each of C. glabrata and C. parapsilosis had high MIC s (>2 mg/ml) to caspofungin, while 10 isolates of C. parapsilosis also had high MIC s to anidulafungin and micafungin. Two isolates of C. dubliniensis had high MIC s (>2 mg/ml) to all the echinocandins. These results are similar to those published by other studies that show the very low MICs typical for wild-type strains of C. albicans, C. glabrata, C. tropicalis, C. krusei and C. kefyr and the higher MICs typical of C. parapsilosis, C. guilliermondii, and C. lusitaniae for the echinocandins. 7,8 All isolates tested were also susceptible to amphotericin B. Resistance to fluconazole was detected in isolates of C. krusei and C. glabrata. The majority of C. glabrata strains and some isolates of Cryptococcus were found to be susceptible dose dependent (SDD) to fluconazole (MICs μg/ml). Resistance to itraconazole (MIC >1 μg/ml) was more widespread and also there were a greater number of isolates in the SDD range (MICs μg/ml). This occurred predominantly in isolates of C. glabrata, C. parapsilosis and C. krusei and in some isolates of Cryptococcus. Voriconazole resistance (MIC >4 μg/ml) was limited ASA Newsletter, August

4 to a few isolates of C. glabrata; all voriconazole resistant C. glabrata stains were cross resistant to fluconazole and itraconazole. Suggested likely resistance to posaconazole (MIC s >2 μg/ml) occurred in 161/303 (53%) of C. glabrata isolates and in 1 isolate of C. tropicalis. These results are in keeping with other studies that show intrinsic resistance to antifungal agents by Candida and Cryptococcus species is largely predictable based on accurate identification of the organism. 9,10 Resistance to amphotericin B and the echinocandins is uncommon, while resistance to the azoles is largely confined to isolates of C. glabrata and C. krusei. 9,10 B. CLSI M44-A2 and M44-S3 disk diffusion standard for yeasts 11,12 The M44-A2 standard and M44-S3 informational supplement is intended for disk diffusion susceptibility testing of Candida species 11,12. The standard includes zone interpretive criteria for caspofungin, fluconazole and voriconazole. It recommends the use of Mueller-Hinton agar supplemented with 0.2% glucose and 0.5 ug/ml methylene blue dye medium. Mueller-Hinton agar is readily available and shows acceptable batch-to-batch reproducibility, the glucose provides a suitable growth for most yeasts and the addition of methylene blue enhances the zone edge definition. The ph of the medium needs to be between 7.2 and 7.4 at room temperature after gelling. The inoculum is standardized to 0.5 McFarland using a densitometer and plates should be incubated at 35 o C for 24 hours. Some strains where insufficient growth has occurred after 24 hours may need to be read after 48 hours incubation. Commercially prepared paper disks for fluconazole (25 ug) and voriconazole (1 ug) are available from Oxoid and Becton Dickinson. Neo-Sensitabs (manufactured by Rosco in Denmark) are available in Australia by Dutec Diagnostics. Neo-Sensitabs are cheap, agar diffusion tests are easy to set up and show potential for the screening of large numbers of isolates for resistance. Susceptibility data using the CLSI M44-A2 disk method representing Candida isolates ( ) recovered from genital sites are shown in Table 3. Table 3: Australian antifungal susceptibility data for Candida isolates from recurrent vulvovaginal candidiasis ( ). Fluconazole Clotrimazole Nystatin S SDD R S I R S I R C. albicans 220 (82%) C. glabrate 35 (13%) C. parapsilosis 4 (1.5%) C. krusei 4 (1.5%) C. tropicalis 3 (1%) C. kefyr 2 (0.7%) C. dubliniensis 1 (0.4%) C. guilliermondii 1 (0.4%) ASA Newsletter, August 2010

5 A total of 270 Candida isolates from patients with a clinical history of recurrent vulvovaginal candidiasis were tested against fluconazole, clotrimazole and nystatin. Candida albicans accounted (82%) and C. glabrata (13%) accounted for 95% of the isolates. Resistance rates for all the agents tested were very low; 10 isolates against fluconazole (7 C. glabtata and 3 C. krusei), 1 C. glabrata isolate against clotrimazole, and 1 C. albicans against Nystatin). All isolates of C. albicans were susceptible to fluconazole and clotrimazole, however as expected 20% of C. glabrata isolates were resistant to fluconazole, although all were susceptible to Nystatin. A previous USA study of patients with suspected vulvovaginal candidiasis reported the incidence of C. albicans as 70% and that for C. glabrata as 19% 14. These authors also reported a low fluconazole resistance rate of 3.7% for C. albicans and a similar expected higher rate of 15.2% for C. glabrata. In conclusion, species identification was predictable of the antifungal susceptibility. 9,10,13 Candida albicans remains the dominant pathogen in vulvovaginal candidiasis and antifungal drug resistance does not appear to be a factor in determining recurrent infection. Finally, the role of non-albicans Candida species in vulvovaginal candidiasis remains uncertain and further studies are warranted. 15 References: 1. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard-Third Edition. CLSI document M27-A3 Wayne, PA: Clinical and Laboratory Standards Institute; CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Third Informational Supplement. CLSI document M27-S3 Wayne, PA: Clinical and Laboratory Standards Institute; Rex JH, Pfaller MA, Galgiani JN et al. (1997). Development of interpretative breakpoints for antifungal susceptibility testing; conceptual framework and analysis of in vitro - in vivo correlation data for fluconazole, itraconazole and Candida infections. Clin Infect Dis. 24: Witt MD, Lewis RJ, Larsen RA, et al. (1996). Identification of patients with acute AIDS-associated cryptococcal meningitis who can be effectively treated with fluconazole: The role of antifungal susceptibility testing. Clin Infect Dis. 22: Espinel-Ingroff A, Pfaller M, Messer SA. et al. (1999). Multicenter comparison of the Sensititre YeastOne colorimetric antifungal panel with the National Committee for Clinical Laboratory Standards M27-A reference method for testing clinical isolates of common and emerging Candida spp., Cryptococcus spp., and other yeasts and yeast-like organisms. J Clin Micro. 37: Pfaller MA, Chaturvedi V, Diekema DJ et al Clinical evaluation of the sensititre yeastone colorimetric antifungal panel for antifungal susceptibility testing of the echinocandins anidulafungin, caspofungin and micafungin. J Clin Microbiol. 46: Pfaller, MA, Boyken L, Hollis RJ, et al In vitro susceptibilities of Candida spp. to caspofungin: four years of global surveillance. J Clin Microbiol. 44: Pfaller, MA, Boyken L, Hollis RJ, et al Wild-type MIC distributions and epidemiological cutoff values for echinocandins and Candida spp. J Clin Micribiol. 48: Pfaller MA and Diekema DJ The epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Reviews. 20: Pfaller MA, Diekema DJ, Gibbs DL, et al Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-Year Analysis of Susceptibilities of Candida to Fluconazole and Voriconazole Determined by CLSI Standardized Disk Diffusion. J Clin Microbiol : CLSI. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeast; Approved Guideline Second Edition. CLSI document M44-A2. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI Zone Diameter Interpretive Standards, Corresponding Minimal Inhibitory Concentration (MIC) Interpretive Breakpoints, and Quality Control Limits for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Informational Supplement. CLSI document M44-S3 Wayne, PA: Clinical and Laboratory Standards Institute; Meis FGM, Petrou M, Bille J, et al A Global Evaluation of the Susceptibility of Candida to Fluconazole by Disk Diffusion. Diagnostic Microbiology and Infectious Disease 36: Richter SS, Galask RP, Messer SA, et al Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases. J Clin Microbiol. 43: Dennerstein GJ, Ellis DH Oestrogen, glycogen and vaginal candidiasis. Aust NZJ Obstet Gynaecol. 41:3:326. ASA Newsletter, August